Screening of Sun Protective Activity of the Ethyl Acetate Extracts of Some Medicinal Plants

Natural substances extracted from plants have recently been considered as potential sunscreen resources because of their ultraviolet ray absorption in the UVA region and their antioxidant activity. In the present study, the UV protective effects of ethyl acetate extracts of some common medicinal plants, which have flavonoids and other phenolics as the most important components, were evaluated using diffuse transmittance method and calculating Sun Protection Factors (SPFs). The ethyl acetate extracts of sixteen medicinal plants were analyzed for their in vitro SPF by measurement of their transmittanceat different concentrations in methanol. Extracts of leaves of Dracocephalum moldavica L. (Lamiaceae) and flowering tops of Viola tricolor L. (Violaceae) had the highest SPFs, i.e.24.79 and 25.69, respectively. D. moldavica and V. tricolor had high amounts of phenolic compounds and flavonoids., which could be the cause for their high SPF.

Copyright ? 2008 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health Services

Original Article

Screening of Sun Protective Activity of the
Ethyl Acetate Extracts
of Some Medicinal Plants

Payam Khazaelia and Mitra Mehrabanib*

aDepartment of Pharmaceutics, Faculty of Pharmacy,
Kerman University of Medical Sciences, Kerman, Iran. bDepartment of
Pharmacognosy, Faculty of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran.

Abstract

Natural substances extracted from plants have recently been considered as
potential sunscreen resources because of their ultraviolet ray absorption in the
UVA region and their antioxidant activity. In the present study, the UV
protective effects of ethyl acetate extracts of some common medicinal plants,
which have flavonoids and other phenolics as the most important components, were
evaluated using diffuse transmittance method and calculating Sun Protection
Factors (SPFs). The ethyl acetate extracts of sixteen medicinal plants were
analyzed for their in vitro SPF by measurement of their transmittanceat
different concentrations in methanol.

While there is little literature on the ways by
which people protected themselves against the sun, evidence from paintings
suggests that clothes covering the body, veils and large brim hats were used by
ancient Greeks, and umbrellas were used in ancient Egypt, Mesopotamia, China and
India. In 1889, Widmark used acidified quinine sulfate to absorb UVB. Apparently
due to the fact that quinine fluoresces when irradiated with UV, he rightly
assumed that it would absorb the short wavelengths. In 1891, Hammer repeated
Widmark?s experiments using quinine prepared in the form of a lotion or ointment
as the first human sunscreen. At the turn of the century, various plant extracts
had been used in folk medicine (1).

Sunscreens are chemicals that provide protection
against the adverse effects of solar and in particular UV radiation (3). Natural
substances extracted from plants have been recently considered as potential
sunscreen resources because of their ultraviolet ray absorption in the UV region
(4) and their antioxidant activity (5.Green tea polyphenols, Aloe barbadencis
extract, aromatic compounds isolated from lichens and several glycosides of
aesculin are examples of natural substances evaluated for their sunscreen
properties (1, 3, 6, 7).

There is strong evidence that DNA-damaging
ultraviolet (UV) light induces the accumulation of UV light-absorbing flavonoids
and other phenolics in dermal tissue of the plant body. This suggests
physiological function, yet speculative in light protection in plants and of
course in human (5, 8). Ethyl acetate could particularly extract flavonoids and
other phenolics from plants (9).

In the present study, we evaluated the UV
protective activities of ethyl acetate extracts of some most available and
common medicinal plants which have flavonoids and other phenolics as the most
important chemicals (10, 11) using diffuse transmittance method and calculating
Sun Protection Factors (SPFs).

Experimental

Plants

Different parts of sixteen medicinal plants were
collected from different regions of Iran. The specimens were prepared and
authenticated (Table 1). The herbal samples were then kept at the herbarium of
Faculty of Pharmacy, Kerman University of Medical Sciences.

Extraction procedure

The air-dried herbal materials were ground into
fine powders and extracted by maceration method two times with 90% and 50%
hydro-methanol solutions respectively (12). After filtration of total extracts,
the solvent was removed under reduced pressure to give the gummy mass. Then
these residues were dissolved in 20% hydro-methanol solution and kept in
refrigerator for 48 h until the chlorophyll was precipitated. The extracts were
filtrated and extracted with ethyl acetate in decanter until the ethyl acetate
was exactly uncolored (9). These extracts were dried under reduced pressure and
the yield values were determined.

Screening of sun protection activity by
determination of the in vitro sun protection factor (SPF)

The ethyl acetate extracts of plants were analyzed
for the in vitro SPF. The extracts were dissolved in methanol UVsolv
(Merck) (13, 14). Scanning spectra of the samples in solution were obtained by
running from 337.5 to 292.5 nm (at five nm intervals). The SPF determination
model used in this study based on the following equation proposed by Gharavi
et al. (15):

where: T(λ)is the measured sunscreen
transmittance at λ; E(λ) is the spectral irradiance of terrestrial sunlight
at λ; and ε(λ) is the erythemal action spectrum at λ.

The values of the E(λ) and ε(λ) were calculated
according to the report by Gharavi et al. (15).

T(λ) was measured four times for each extract and
the mean was used for calculation of SPF. Three different concentrations of each
extract (according to Beer?s Law) were used for obtaining the standard curve and
calculating SPF in 2 mg/ml solution according to Gharavi et al. method
(15).

Results and discussion

Table 1 shows the SPFs of 2 mg/ml in solutions of
extracts methanol. Extracts of leaves of Dracocephalum moldavica L. (Lamiaceae)
and flowering tops of Viola tricolor L. (Violaceae) had the highest SPFs,
i.e.24.79 and 25.69 respectively. SPFs of methanol solutions of flowers of
Calendula officinale L. (Asteraceae) and flowering tops of Hypericum
perforatum L. (Hypericaceae) were 12.01 and 12.21 respectively. Sun
protective activities of the rest of the plants were low representing by their
sun protection being lower than 10.

In this study, among the sixteen plants studied,
only Dracocephalum moldavica and Viola tricolor had sun protective
activity, considering the SFP values actors of higher than 20.

Most spectrophotometric techniques for
transmittance measurements rely on preparing samples with a uniform and known
thickness so that the optical path length through the sample is standardized.
Many samples are dissolved in special solvents and placed into 10 mm path length
cuvettes. The cuvettes for UV spectrophotometry are usually made from quartz
(fused silica) which is transparent to UV wavelengths (14).

In the Solvent method, different concentrations of
test products in methanol were prepared. Each sample?s transmittance was
measured to evaluate the SPF value.

Sunscreens contain a wide variety of chemicals that
have specific absorbance in some parts of the UV spectrum. There are very few
single chemical substances that have absorbance over the full range of UV. This
property is needed for a product to be considered as a proper wide-spectrum
sunscreen. Plant extracts, due to containing a wide range of natural compounds,
usually cover this full range of UV warelengths. One approach to protecting the
body from the harm?ful effects of UV irradiation is to use active
photoprotec?tives. In recent years, naturally occurring compounds have gained
considerable attention as protective agents (16).

There is a review about the pho?toprotective
effects of some naturally occurring herbal polyphenols and phenolic- rich
extracts in studies of the skin damage induced by UV irradiation. Several
studies have shown the flavonoids to act as scavengers of superoxide anions,
singlet oxygen, hydroxyl radicals, and lipid peroxyl radicals. There are also
reports of flavonoids inhibiting the activities of many enzymes, including
lipoxygenase, cyclooxygenase, monooxygenase, xantinoxidase, mitochondrial
succinate dehydrogenase and NADH-oxidase, phospholipase A2, protein kinases, and
nuclear transcription factor. Phenolics are believed to be capable of acting in
redox-sensitive signalling cascades to inhibit DNA damage. In contrast to their
beneficial effects, some phenolics however have also been found in vitro
to be mutagenic. These harmful effects are suspected to result from the
prooxidant rather than antioxidant action of these com?pounds. Therefore, it is
necessary to investigate their toxic effects before human use. In the past
decade, the antioxidant activity of herbal phenolics, namely phenolic acids and
flavonoids, has been given much attention. Many flavonoids such as quercetin,
luteolin and catechins are better antioxidants than the nu?trients vitamin C,
vitamin E and β-carotene. Therefore, the phenolics may be beneficial in
preventing UV-induced oxygen free radical generation and lipid peroxidation,
i.e. events involved in pathological states such as photoaging and skin cancer
(16).

Topical application of extract of flower buds of
Capparis spinosa reduces UV-induced skin erythema in healthy human
volunteers. Topical application of Culcitium reflexum extract in the form
of a gel has proven to be a significant in vivo protection against
UV-induced skin erythema in healthy human volunteers. Ginkgo biloba
extract has also been found to decrease the number of UVB-induced sunburn cells
in mice skin. Oral administration of G. biloba extract leads to increased
super-oxide dismutase activity in UVB-irradiated mice skin. Extract of
Krameria triandra root has shown protective effects against UVB-induced
photodamage in human keratino?cyte cells. The extract of Prunus persica
flower was also shown to delay tumour development after UVB-induced skin
carcinogenesis in SKH-1 hairless mice. It also reduces UVB-induced ear edema in
IRC mice. The lyophilised flavonolic frac?tion of Sedum telephium leaf
extract appears to possess potent protective effects against UV-induced skin
erythema in human volunteers (16).

Dracocephalum moldavica is an annual plant
native in Central Asia and naturalized in Eastern and Central Europe where it is
used in folk medicine as antiseptic and stimulating remedy. It contains high
amounts of phenolic compounds such as: rosmarinic acid: 2.84 g%, caffeic acid:
50.3 mg%, total flavonoids: 0,58 g% ( apigenin, luteolin flavon aglycons), and
tannins: 12.9 g% (18).

Viola tricolor is an annual plant indigenous
to Eurasia rigon. It is traditionally used for bronchitis and rheumatism. V.
tricolor is an especially valued remedy for treating skin diseases. Used
both internally and topically, it is good for eczema, psoriasis and acne. The
active compounds are flavonoids, including violanthin, rutin, violaquercitrin
and salicylates. Both salicylates and rutin contained in the plant are anti-inflammatoryagent.
Due to the high concentration of rutin in the flowers, thisplant may be used to
prevent UV-induced oxygen free radical generation (19, 20).

D. moldavica and V. tricolor had high
amounts of phenolics and flavonoids. The high value of SPF (24.79 and 25.69
respectively) could be due to these chemicals.

For following studies, it is suggested to prepare
topical sunscreen formulations from these two extracts and measure their SPFs.

Acknowledgement

This work was supported by a grant from the
Research Council of Kerman University of Medical Sciences, Kerman, Iran.. We
woul like to thank Dr. S. Derafsh, Dr. A. Abedini, Dr. Sh. Ahadpour and Dr. S.M.
Majd-zadeh for the technical works.